Prolonged exposure to decreased oxygen tension, as occurs in many pulmonary diseases, results in pulmonary hypertension (CHPH). Emerging evidence indicates sustained pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be related to changes in K channels, membrane potential and intracellular Ca2+ concentration ([Ca2+]i); however, the exact mechanisms underlying, and factors mediating, this process remain unknown. Hypoxia-inducible factor 1 (HIF-1), a transcription factor, mediates numerous adaptive responses to hypoxia. HIF-1 controls hypoxic induction of many genes that may be important in development of CHPH, including endothelin- I (ET-12). ET-1 receptor antagonists prevent CHPH and ET-1 inhibits CH. Furthermore, in PASMC from chronically hypoxic animals, ET-1 signal transduction pathways are altered such that ET-1 -induced contraction is enhanced and appears to occur primarily via activation of Ca2+ -independent contractile pathways, possibly due to activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Enhanced contraction coupled with elevated ET-1 levels could result in a positive feedback mechanism by which sustained vasoconstriction is maintained. We developed a murine model of CHPH that, together with the generation of transgenic mice with partial deficiency for the varies as subunit of HIF-1, creates a unique animal model system that allows us to specifically target the role of HIF-1 in this disease process. We will use this model to test the hypothesis that during CH, induction of HIF-1 is a critical initiating step in the development of CHPH, and that hypoxic induction of HIF-1 results in elevated ET-1 levels which activate a combination of contractile mechanisms in PASMQs, including: a) depolarization due to reduction of K+ channels; b) depolarization-driven elevation of resting [Ca2+]i and c) changes in Ca2+ -sensitivity of the contractile apparatus due to activation of PKC- and MAPK- dependent pathways. To test this hypothesis, we will use a combination of techniques, including isometric tension recording and Western blot analysis in arterial segments, and whole-cell patch-clamp and microfluorescence measurement in PASMCs, to accomplish the following Specific Aims: 1) determine whether HIF-1 regulates ET-1 levels and mediates CH-induced alterations observed in PASM; 2) determine whether ET-1 mediates CH-induced alterations observed in PASM; 3) determine whether CH decreases K+ channel protein expression and whether this is regulated by HIF-1 and ET-1; 4) determine whether the CH-induced elevation in resting [Ca 2+]i is due to depolarization-driven enhancement of Ca2+ influx through Na+/Ca2+ exchange and 5) determine the role of PKC and MAPK in ET-1 -induced increase in Ca2+ sensitivity in PASM following exposure to CH. (End of Abstract)